Int6 has been implicated in the development of breast cancer because it was first identified from a genetic screen in which the mouse mammary tumor virus (MMTV) was used as an insertional mutagen. MMTV insertions in int6 lead to C-terminal truncations in the protein, thus removing its PCI domain (Proteasome-COP9-lnitiation factor), which is found frequently among proteins that interact with the 26S proteasome. With the support of our current grant, we have investigated the molecular functions of Int6 by using a genetic model system the fission yeast Schizosaccharomyces pombe. Our data support a hypothesis in which Int6 regulates mitotic abnormalities and chromosome segregation by controlling the proper assembly and localization of the proteasome. We propose that Int6 acts as a tumor suppressor. When Int6 is inactivated, the proteasome becomes inactive, thus leading to the accumulation of mitotic regulators blocking proper cell division control and chromosome segregation. In this renewal, our goals are to further define how Int6 controls proteasome assembly and localization in the S. pombe system and to examine whether the functions of Int6 as revealed in S. pombe can also function in humans and play roles during tumorigenesis. Our Aim 1 is to determine whether S. pombe Int6 can allow the proteasome to more selectively and efficiently degrade given substrates by acting through another evolutionary conserved protein called Moe1, which binds proteins such as Cdc48 and APC (Anaphase Promoting Complex) that are known to bind polyubiquitinated proteasome substrates. In Aim 2 we will study how Int6 influences proteasome localization by examining the possibility that Int6 in S. pombe regulates the F-actin cytoskeleton via a component of the Arp2/3 complex called Arc21, which may mediate the proper formation of transport vesicles. In Aim 3, we will directly examine in human mammary epithelial cells whether Int6 also regulates the proteasome to influence mitosis and chromosome segregation and whether its inactivation can transform these cells and form tumors in nude mice. We believe that understanding the regulation of the proteasome by Int6 can lead to the design of better cancer diagnosis and more focused cancer therapies.